PROJECT SUMMARY/ABSTRACT Eukaryotic genome structure fluctuates across both 3-dimensional space and time and must be precisely orchestrated to achieve regulated gene expression. This structure underlies regulatory interactions between distal control elements (i.e. enhancers) and the genes that they target. While many thousands of enhancers have now been annotated and their interacting genic partners identified, we have a poor understanding of the molecular nature of these interactions, their regulation, and their significance in the cell. Chromosome conformation capture (3C) techniques have accelerated study of chromosomal organization but provide only population-averaged snapshots with poor temporal resolution and so fail to describe chromosomal interactions in their relevant context. Elucidating the molecular basis of enhancer-gene interactions in living, single cells is paramount to understanding transcriptional control. To address this need we are building new tools to visualize and manipulate chromosomal interactions in living cells. This proposal aims to dissect enhancer function by probing the dynamics and control of a model chromosomal interaction: the Sox2 gene and its distal, essential Sox2 Control Region (SCR). Sox2 encodes a tissue-specific transcription factor (TF) involved in pluripotency and reprogramming, making its transcriptional control of broad interest. In mouse embryonic stem cells (ESCs), Sox2 expression is established through the function of the SCR, located >100 kb away. To probe the dynamics of Sox2's interaction with its enhancer in living ESCs, we are combining techniques for marking DNA with advances in genome editing. To determine how these dynamics inform gene expression, we are developing tools to acutely and specifically manipulate the stability of the Sox2/SCR interaction and assay the consequences on Sox2 gene expression in individual living cells. These studies should broadly inform our understanding of transcriptional control by enhancer elements and should be generalizable to a wide range of other genomic contexts.